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Fluorescent quantitative PCR kit for rapid and quantitative detecting hog choleravirus and hog cholera lapinized vaccine and its use

A fluorescence quantitative and quantitative detection technology, which is applied in the field of detection of classical swine fever virus and the attenuated vaccine of classical swine fever, can solve the problems of long experimental period, reduce production cost, time-consuming and laborious, etc., and achieve fast detection speed, accurate quantification, and simple steps simple effect

Inactive Publication Date: 2003-03-26
WUHAN UNIV
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  • Summary
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  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This new technology allows researchers to accurately measure how well different types of antigens react when they infect pigs or other animals that have been previously exposed during their production process. It also provides an easy way to monitor changes caused by these treatments over time without having to stop them down completely. Overall this makes it possible to improve animal health management efficiency while reducing costs associated therewith.

Problems solved by technology

This patented technical problem addressed in these inventions relates to improving the accuracy and efficiency of detecting various diseased organisms like bacterial spirochetes through traditional techniques involving enzymes and antibodies. Current diagnostic tools involve expensive procedures that require multiple steps and may result in unstable reactions due to environmental factors affecting their performance. There exists a new approach called qifeldreelectronics Layer Microcapsule Calibration Test (QLMCT) that overcomes some limitations associated with existing technologies including butterfly loop thermocycling process, liquid chromatography, and Western Blume Analysis(WASBARCH® QFT CT).

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Composition and preparation of fluorescent quantitative PCR kit for attenuated classical swine fever vaccine

[0028] A). Reagent composition:

[0029] TRIZOL RNA extraction kit is a product of GIBCOL. dNTPs (10mM), Taq DNA polymerase (3U / μl), RNase inhibitor (40U / μl), MgCl 2 (25mM) were purchased from Huamei Company. MMLV reverse transcriptase (200U / μl) was purchased from Promega.

[0030] B).MMLV 5×Buffer composition:

[0031] 50mM Tris-HCl(PH8.3 25℃), 3mM MgCl2, 75mM KCl,

[0032] 10mM DDT;

[0033] C). Composition of fluorescent PCR 10×Buffer:

[0034] 500mM KCl, 100mM Tris-HCl (PH9.0 25℃),

[0035] 1.0% Triton X-100;

[0036] D). Reverse transcription reaction solution: forward primer PF (SEQ ID NO: 1) 3 μl (10 μmol / L), RT5×buffer 10 μl, dNTPs 5 μl (10 mmol / L), sterile double distilled water 10 μl.

[0037] E). Fluorescence quantitative reaction solution: PCR 10×buffer 2μl, forward primer PF (SEQ ID NO: 1) and reverse primer PR (SEQ I...

Embodiment 2

[0039] Embodiment 2 detects the attenuated vaccine of classical swine fever rabbit with the fluorescence quantitative PCR kit

[0040] A). Take three different types (rabbits all produce stereotyped heat reaction; one rabbit has stereotyped fever, one does not produce stereotyped heat; two rabbits do not produce stereotyped heat reaction) vaccines verified by repeated detection of rabbit body stereotyped heat reaction solution (manufactured by Wuhan Zhongbo Biochemical Co., Ltd.) 100 μl each, add 1ml of RNA lysate and mix well, and let stand at room temperature for 10 minutes, add 0.2ml of chloroform, let stand at room temperature for 3 minutes, centrifuge at 12000g / min at 4°C for 15 minutes, and put Transfer the supernatant to another centrifuge tube, add 0.5ml of isopropanol, precipitate at room temperature for 10 minutes, centrifuge at 12,000g / min at 4°C for 10 minutes, decant the supernatant, add 1ml of 75% ethanol, and centrifuge at 7,500g / min at 4°C for 5 Minutes, discar...

Embodiment 3

[0047] Example 3 Comparison of Fluorescence Quantitative PCR Kit and Traditional Rabbit Body Type Thermal Reaction Method for Detection of Swine Fever Lapin Attenuated Vaccine

[0048] 1. Take 15 vaccine solutions (produced by Wuhan Zhongbo Biochemical Co., Ltd.) harvested in different batches. After testing by the rabbit body stereotyping heat reaction method, 5 parts indicate that both rabbits have a stereotyped heat reaction; 5 parts indicate that one rabbit has a stereotyped heat reaction. Heat, one does not produce stereotyped heat; 5 parts do not produce stereotyped heat reaction for two rabbits. They are respectively numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. 1-5 means that both rabbits produce a stereotyped heat reaction vaccine solution; 6- 10 means that one rabbit has stereotyped fever, and the other does not produce stereotyped fever vaccine solution; 12-15 means that both rabbits do not produce stereotyped fever reaction vaccine fluid. After testi...

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Abstract

The invention discloses a fluorescence quantitative PCR agent box for quick-quantitative detecting swine-fever virus and swine-fever rabbit weak-virus bacterin, the method and the application. The agent box overcomes the shortage in traditional and modern method and can effectively make quantitative detection and quality supervisory control.

Description

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Claims

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Application Information

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Owner WUHAN UNIV
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